Study On The Metabolic Toxicity,Susceptibility And Mechanism Of DEHP On Adolescent Mice With Type 2 Diabetes Mellitus(T2DM)

Di（2-ethylhexyl）phthalate（DEHP）,one type of widely distributed phthalates in the environment,has been detected in blood,urine and other biological samples of humans and is harmful to human health.Adolescents are suffering from DEHP exposure of higher levels than adults.DEHP exposure could lead to obesity,insulin resistance and metabolic disorders.Currently,the effects of DEHP exposure on the health of adolescent type 2 diabetes mellitus（T2DM）attracted worldwide concern.In this paper,the insulin resistance BRL cells and adolescent ICR mice were exposed to DEHP to investigate the metabolic toxicity of low dose DEHP.Based on the above experiments,the models of adolescent T2DM mice were established to investigate the metabolic toxicity and susceptibility of DEHP on adolescent T2DM mice.And then the molecular mechanisms were clarified.This research could provide scientific basis for protecting the health of adolescent T2DM.The main conclusions of this study are given below:（1）Study on the metabolic toxicity of DEHP on insulin resistance BRL cells.It was discovered that the IC₅₀ of DEHP on insulin resistance BRL cells was significantly higher than that on normal BRL cells.The results showed that low dose DEHP significantly increased the ALT and AST levels in the culture of insulin resistance BRL cells and increased the Caspase-3 activity in insulin resistance BRL cells,which demonstrated that DEHP exposure of low dose could impair insulin resistance BRL cells,and induce apoptosis.Besides,DEHP could significantly inhibited the expressions of insulin receptor,IRS-1,GLUT4,PI3K,AKT and their related genes,which lead to the decreased insulin sensitivity and glucose transport.Through factorial analysis,there were synergistic effects between insulin resistance and DEHP exposure on the metabolic functions of BRL cells.Therefore,DEHP exposure could induce metabolic toxicity to insulin resistance BRL cells,which was more significant than that to normal BRL cells.The potential mechanism is that DEHP exposure leads to the metabolic toxicity to insulin resistance BRL cells through disturbing insulin receptor signaling pathway.（2）Study on the metabolic toxicity of DEHP on adolescent ICR mice.The results showed that DEHP could significantly increase the serum Hb A1c level,which disturbed the glucose metabolism in adolescent ICR mice.DEHP significantly increased the serum levels of TC,LDL and TG,and decreased the levels of serum HDL,serum LCAT and hepatic HL in the adolescent ICR mice,which lead to the disorders of lipid metabolism in adolescent ICR mice.It was discovered that DEHP could significantly affected the expressions of insulin receptor,IRS-1 and GLUT4 as well as the IRS-1phosphorylation in the pathways of insulin sensitivity and glucose transport in the adolescent ICR mice.Factorial analysis revealed that the adolescent and DEHP exposure had the synergistic effects on the lipid metabolism,insulin sensitivity and glucose transport of adolescent ICR mice.Therefore,DEHP could induced metabolic toxicity to adolescent ICR mice,which was more sensitive than adult ICR mice.The potential mechanism is that DEHP could disturb the pathways of insulin sensitivity and glucose transport in the adolescent ICR mice to induce metabolic toxicity.（3）Study on the metabolic toxicity,susceptibility and molecular mechanism of DEHP on the male adolescent T2DM ICR mice.When compared with untreated male adolescent T2DM（A-T2DM）mice,DEHP exposure could significantly disturb the glucose metabolism and lipid metabolism of male A-T2DM.The levels of FBG and HOMA-IR and the serum levels of insulin,C-peptide,TC,TG and LDL in male A-T2DM mice were significantly increased by DEHP exposure,while the serum LCAT level and the levels of glycogen,G6PD,GCK and HL in the liver were significantly decreased by DEHP.It could be discovered that DEHP induced metabolic disorders in male A-T2DM mice.Hepatic transcriptome showed that DEHP could significantly affected the gene expressions of male A-T2DM mice,and 293 DEGs were enriched.Through GO analysis,113 DEGs related to metabolic functions were enriched.The KEGG analysis showed that the DEGs could be enriched in Amino acid metabolism,Energy metabolism,Lipid metabolism,Carbohydrate metabolism,Endocrine pathways,Digestive systems,etc.Through further analysis on the transcriptomic data,it was discovered that DEHP could interfere with the feedback regulation of metabolic system and endocrine system in male A-T2DM mice.Western-blot results showed that DEHP could significantly affected the related protein expressions and phosphorylation of insulin receptor,IRS-1,GLUT4,PI3K,AKT,GSK-3β,m TOR,SHC and ERK1/2 in insulin receptor signaling pathway in male A-T2DM mice to induce metabolic toxicity.The analysis on the susceptibility revealed that male A-T2DM mice were more sensitive to the metabolic toxicity of DEHP than the male adolescent normal mice.It could be seen that DEHP had metabolic toxicity to male A-T2DM mice,which was more significant than male adolescent normal mice.The potential mechanism is that DEHP leads to metabolic toxicity via inhibiting the feedback regulation of endocrine and metabolic system and disturbing the insulin receptor signaling pathway（4）Study on the metabolic toxicity,susceptibility and molecular mechanism of DEHP on the female adolescent T2DM（A-T2DM）mice.DEHP exposure of low dose significantly disturbed the metabolism of glucose and lipid in female A-T2DM mice to lead to metabolic toxicity.However,the metabolic toxicity of DEHP exposure to female A-T2DM mice was different from male A-T2DM mice.DEHP could significantly increase the secretions of insulin and C-peptide in female A-T2DM mice,which was contrary to male A-T2DM mice.The analysis on susceptibility showed that the metabolic toxicity of DEHP to female A-T2DM mice was more significant than that to female adolescent normal mice and male A-T2DM mice.Hepatic transcriptome showed that 220 DEGs were enriched in female A-T2DM mice after DEHP exposure,and 66DEGs related to metabolic functions were enriched though GO analysis.KEGG analysis showed that the DEGs were mainly enriched in Apoptosis,MAPK signaling pathway,Drug metabolism,Retinol metabolism,Chemical carcinogenesis,and Type 2diabetes mellitus.Through Western-blot experiment,it was discovered that DEHP could affect the protein expressions and phosphorylation of JNK,Caspase-3,BAX,Bcl-2 and ERK1/2 to further disturb the protein expressions and phosphorylation of insulin receptor,IRS-1,GLUT4,PI3K,AKT,GSK-3βand m TOR in insulin receptor signaling pathway,which exacerbated the insulin resistance and metabolic disorders of female A-T2DM mice.It could be concluded that DEHP induced metabolic toxicity to female A-T2DM mice,which was more significant than female adolescent normal mice and male A-T2DM mice.The mechanism for DEHP-induced metabolic toxicity to female A-T2DM mice was different from that to male A-T2DM mice.DEHP exacerbated the metabolic disorders of female A-T2DM mice via inducing oxidative impairment in liver and then inhibiting insulin sensitivity.